Trace glucose and lipid metabolism in high androgen and high-fat diet induced polycystic ovary syndrome rats.

Zhai et al. Reproductive Biology and Endocrinology 2012, 10:5
http://www.rbej.eom/content/1 0/1 /5

Airrjr'-; REPRODUCTIVE BIOLOGY
Ja_^>^_. and endocrinology

RESEARCH

Open Access

Trace glucose and lipid metabolism in high
androgen and high-fat diet induced polycystic
ovary syndrome rats

Hua-Ling Zhai, Hui Wu, Hui Xu, Pan Weng, Fang-Zhen Xia, Yi Chen and Ying-Li Lu*

Abstract

Background: There is a high prevalence of diabetes mellitus (DM) and dyslipidemia in women with polycystic
ovary syndrome (PCOS). The purpose of this study was to investigate the role of different metabolic pathways in
the development of diabetes mellitus in high-androgen female mice fed with a high-fat diet.

Methods: Female Sprague-Dawley rats were divided into 3 groups: the control group(C), n = 10; the andronate-
treated group (Andronate), n = 10 (treated with andronate, 1 mg/100 g body weight/day for 8 weeks); and the
andronate-treated and high-fat diet group (Andronate+HFD), n = 10. The rate of glucose appearance (Ra of
glucose), gluconeogenesis (GNG), and the rate of glycerol appearance (Ra of glycerol) were assessed with a stable
isotope tracer. The serum sex hormone levels, insulin levels, glucose concentration, and the lipid profile were also
measured.

Results: Compared with control group, both andronate-treated groups exhibited obesity with higher insulin
concentrations {P < 0.05) but similar blood glucose concentrations. Of the two andronate-treated groups, the
andronate+HFD group had the most serious insulin resistance (IR). Estrus cycles were completely acyclic, with
polycystic ovaries and elevated serum lipid profiles in the andronate+HFD group {P < 0.05). Ra of glucose and GNG
increased significantly in the andronate+HFD rats. However, the Ra of glycerol was similar in the three groups.

Conclusions: Andronate with HFD rat model showed ovarian and metabolic features of PCOS, significant increase
in glucose Ra, GNG, and lipid profiles, as well as normal blood glucose levels. Therefore, aberrant IR, increased
glucose Ra, GNG, and lipid metabolism may represent the early-stage of glucose and lipid kinetics disorder, thereby
might be used as potential early-stage treatment targets for PCOS.

Keywords: Andronate, Glucose metabolism, Lipid metabolism, High-fat diet, Polycystic ovary syndrome

Background

Polycystic ovary syndrome (PCOS) is one of the most
common endocrine disorders in women of reproductive
age [1] and is the most frequent cause of hyperandro-
genism and anovulation [2]. PCOS is also strongly asso-
ciated with abdominal obesity, hyperinsulinemia, insulin
resistance, and type 2 diabetes [3]. The pathophysiology
of PCOS is largely unknown but has been attributed to
defects in various organ systems. Uncontrolled ovarian
steroidogenesis with a thickened thecal layer that secrets

* Correspondence: Iuy662011@yahoo.com.cn

Endocrinology and Metabolism Research Institute and Department of
Endocrinology and Metabolism, Shanghai Ninth People's Hospital Affiliated
Shanghai Jiaotong University School of Medicine, Shanghai 200011, China

excessive androgen is thought to be a primary abnorm-
ality of PCOS [4]. PCOS is combined with defects in
insulin action and insulin resistance (IR) finally leading
to diabetes, and it also displays neuroendocrine dysfunc-
tion with exaggerated LH pulsatility, and altered produc-
tion of adrenal androgen [5].

Once a diagnosis of PCOS is confirmed, it is impera-
tive to assess the woman for diabetes mellitus (DM) risk
factors. Despite the many reasons that women seek
medical care for PCOS, the greatest long-term risk for
these women is DM [6]. The link between PCOS and
DM is multi-faceted [7]. Insulin resistance (IR) is
increased in age-matched PCOS women and is linked to
hyperandrogenism [8]. No single blood test is available

o

© 201 2 Zhai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
BiolVlGCl C6ntTcll Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Zhai et al. Reproductive Biology and Endocrinology 2012, 10:5
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to predict or measure this DM risk. Although no con-
sensus recommendation for the assessment of DM risk
factors exists, measurement of glucose metabolism, lipid
screening, and measurements of insulin concentrations
have been suggested [9].

Due to the heterogeneity of PCOS, it is difficult to
create a single animal model that expresses the main
PCOS characteristics [10]. We used andronate (testos-
terone propionate), a steroid hormone of the androgen
group, in combination with high-fat diet (HFD) to estab-
lish a rat model of PCOS. The aim of this study was to
investigate the metabolic pathways in glucose metabo-
lism, lipid production, and gluconeogenesis with a stable
tracer method. This method allows noninvasive detec-
tion of key steps in glucose and lipid metabolism, lead-
ing to the understanding of the mechanisms by which
PCOS modifies glucose and lipid metabolism and treat-
ment targets for PCOS.

Methods

Animals and experiment design

Female Sprague-Dawley rats (3 weeks old) were bred
and housed locally at 22°C ± 2°C under a 12 h on, 12 h
off light cycle with free access to food and water. The
rats were randomly divided into three groups for the
next 8 weeks of treatment. The protocol of the study is
presented in Figure 1.

Control group (n = 10): The rats were fed with a stan-
dard laboratory diet (52% carbohydrate, 22.1% protein,
9.2% water, 5.28% fat, 4.12% cellulose, 4.22% mineral
salts) and were injected with olive oil of a similar
volume as the experimental group.

Andronate group (n = 10): The rats were injected with
andronate subcutaneously at a dose of 1 mg/100 g body
weight/day for 8 weeks. The rats were also fed with a
standard laboratory diet.

Andronate combined high-fat diet group (n = 10): The
rats were injected with andronate subcutaneously at a

dose of 1 mg/100 g body weight/day for 8 weeks. The
rats were also fed with a lipid-enriched diet (54.2% stan-
dard diet, 16.8% lard, 15% sucrose, 9% casein, 1% miner-
als, 1% vitamins, 3% malt dextrin).

Body weight and blood glucose were measured weekly.
All the animal procedures were performed in accor-
dance with the ethical principles in animal research
adopted by the Department of Laboratory Animal
Science, Jiaotong University School of Medicine, Shang-
hai, China.

Vaginal smears

A moistened cotton bud swab was gently inserted into
the vagina. The cells were removed from the vaginal
lumen and walls and then transferred onto a glass slide.
It is the quickest way of smearing and the smears retain
their original appearance indefinitely. The stage of cycli-
city was determined by microscopic analysis of the pre-
dominant cell types in vaginal smears. The vaginal
smears were analyzed daily since 8 weeks of age to the
end of the experiment.

Ovarian morphology

Ovarian tissue from each group was fixed in 4% parafor-
maldehyde, dehydrated with ethanol and xylene,
embedded in paraffin, and sliced into 5-(im sections on
a microtome (SLEE, Germany). The sections were
stained with hematoxylin-eosin (HE) and analyzed
under an optical microscope (CKX41, Olympus, Japan).

Blood sample collection and assays

One week prior to the treatment, tail blood was
obtained after an overnight fast to assess the glucose,
insulin, and sex steroid levels and the lipid profile.
Plasma glucose and lipids concentration were assayed by
Siemens Dimension MAX (Siemens Healthcare Diagnos-
tics Inc). Plasma insulin was assayed by magnetic affinity
immunoassay (Insulin MPAIA Kit). Sex steroids,

3wk

Swk

Tail blood draw for measurement
of steroids, lipids, FPG,FINS

\

9wk 10wk llwk
i i

vaginal smear every day

experiment conducted: control group, andronate group, andronate+HF diet group

study ended with isotope infusion experiment
and tissues as ovaries was obtained

Figure 1 Research protocol of the experiment. The rats were divided into three groups. The arrows indicate the times of blood draws and
isotope infusion. A vaginal smear was administrated everyday since 8 weeks.

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including follicle-stimulating hormone (FSH), luteinizing
hormone (LH), and testosterone (T) were assayed by
chemiluminescent microparticle immunoassay (CMIA).

Stable isotope infusion procedure

At 11 weeks of age, the tail-artery was catheterized for
blood collection after an overnight fast. During the pro-
cedure, only the tail was anesthetized locally with lido-
caine. The procedure took 15 min to complete. This
catheterization allowed frequent collection of arterial
blood samples while tracers were infused intravenously,
as well as the adherence to the optimal V-A mode of
metabolic experiments. The animals were relaxed with
the ability to move partially, groom and drink. Thus,
experimental stress was minimized. When blood glucose
returned to baseline (usually within 30 min) shown by a
hand-held glucose device (Terumo, medisafe mini blood
glucose reader, manufactured by: Terumo corporation,
Tokyo, Japan), a flexible plastic intravenous infusion line
was placed in the lateral tail vein by venipuncture, and
isotopic tracer infusions were commenced as described
below. The experimental setting is shown in Figure 2.

In vivo experiments

In this study, appearance of glucose and glycerol were
traced by [6,6-2D] -glucose and [U-13 C] -glycerol, while
gluconeogenesis (GNG) were accessed by [1,2,3-13 C] -glu-
cose and [U-13 C] -glycerol, [6,6-2D] -glucose (2 [imol/kg/

min) and [U-13 C] -glycerol (0.84 (imol/kg/min) were
infused intravenously constantly, without priming, through
the tail infusion line driven by an infusion pump (Harvard
Apparatus, Holliston, MA, USA)for 90 min. This is
defined as the basal period. During the last 10 min (80-90
min), arterial blood samples (0.5 ml each) were collected 5
min intervals from the tail arterial catheter. These samples
were used for the quantitation of steady state glucose and
glycerol metabolism. The rats were then euthanized by
heart-opening under anesthesia with pentobarbital (50
mg/kg) in order to reduce blood elements in tissues. Ovar-
ies were harvested swiftly. Plasma was prepared on site
and saved at -80°C for later analysis. A flow chart of the
study design is shown in Figure 3.

Gas chromatography/mass spectrometry

Plasma samples from the infusion experiments were pro-
cessed chromatographically by methoxyamine-HCl and
BSTFA to get m/z: 321/319 [6,6-2D] -glucose, and m/z:
221/218 [U-13 C] -glycerol. To exclude the interference of
spill over from the [6,6-2D] -glucose to [1,2,3-13 C] -glu-
cose (formed in gluconeogenesis whose precursor is [U-13
C] -glycerol), hydroxylamine hydrochloride was used to
derivation. Then the derivation was analyzed by gas chro-
matography/mass spectrometry (autoSystem XL GC/Tur-
boMass MS) for the enrichment. The GC oven
temperature was programmed initially at 70°C for 4 min,
increased to 240°C at 10°C/min, then to 300°C at 20°C/

Figure 2 Experimental setting for stable isotope infusion. The rats catheterized in the tail artery for blood collection
isotope infusion. The rats were relaxed, with the ability to move partially, groom and drink. Thus, experimental stress was

and in the tail vein for
minimized.

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Blood draw

Cathetering
J

Blood draw-

* \ \

-20' 0'
Basal

o\e5-d2-gliicose 2uiriol/l^miti

U- 1 3C-glycenol 0 .S4 uiriol/^iriiti

t

Tissue sampling

Figure 3 Protocol of in vivo infusions of stable isotopes. This is the protocol of steady-state isotope infusion. The arrows indicate the times
for blood draws and tissue sampling.

min, and stayed at 300°C for 11 min. The GC was electro-
nically controlled for constant pressure and humidity.

Calculation of glycerol and glucose appearance rates and
rates of gluconeogenesis

The infusion rate ((imol/kg/min) of [U-13 C] -glycerol
and [6,6-2D]-glucose was divided by mole percent
excess (MPE) of plasma glycerol and that of glucose,
respectively, to give the appearance rates ((imol/kg/min).

Gluconeogenesis rates were calculated by dividing the
MPE of plasma [U-13 C]-glycerol by the MPE of [1,2,3-
13 C] -glucose. In this experiment, appreciable amounts
of [U-13 C] -glycerol were converted to [1,2,3-13 C] -glu-
cose via gluconeogenesis. Thus gluconeogenesis rates
can be compared among different groups.

Statistical analysis

The data were expressed as mean ± standard deviation
(SD), unless otherwise indicated. Statistical significance
was tested by analysis of variance (ANOVA). A value of
P <0.05 was considered significant.

Results

Body weight and blood glucose curves

Andronate-treated two groups exhibited a significant
increase in the mean body weight at the end of the
study (andronate group: 244.5 ± 11.5, andronate + HFD
group: 253.8 ± 19.7; vs. control group: 214.7 ± 13.0, P <
0.05) (Figure 4A). However, there was no significant dif-
ference in blood glucose levels between the three groups
over the 8 weeks time course (Figure 4B).

Reproductive cycles of rats

Reproductive cycles were assessed by vaginal smears.
The control group exhibited regular estrous cycles (Fig-
ure 5), whereas the two experimental groups had pro-
longed or irregular estrous cycles, most of which
showed a constant diestrus smear (Figure 5C).

Ovarian morphology

Light microscope revealed normal ovarian structures in
the control group (Figure 6A, D), with follicles and

corpora lutea in various stages and no ovary cysts were
observed. The ovaries of the andronate-treated group
were much smaller than those of the control group (Fig-
ure 6B), with many cystic follicles with apoptotic granu-
losa cells (Figure 6E). The andronate + HFD group had
larger ovaries than the control group, with a fatty infil-
tration surrounded the ovaries (Figure 6C). In addition,
cystic follicles with macrophages and fluid were also
observed (Figure 6F). In general, both experimental
groups had fewer corpus lutea and preovulatory follicles
and had more preantral and antral follicles than the
controls.

Body hair changes

After 8 weeks treatment with andronate and diet chan-
ging, the two treated groups showed hirsutism and
longer body hair, while the control group had no unu-
sual body hair changes (Figure 7).

Fasting blood glucose (FBG) levels, fasting plasma insulin
(FINS) levels, homeostasis model-insulin resistance
(HOMA-IR), and insulin sensitivity index (ISI)

The FBG levels were similar among all the three groups,
approximately 5 mmol/L. The FINS concentrations were
10.0 ± 1.1 mU/L (control), 15.9 ± 2.1 mU/L (andronate),
and 20.3 ±1.8 mU/L (andronate and HF), respectively.
The HOMA-IR in the andronate + HFD group was
markedly elevated (4.43 ± 0.45), while was mildly ele-
vated in the andronate group (3.49 ± 0.49) compared to
the control group (2.14 ± 0.21). The insulin sensitivity
index was inverse, as both the andronate + HFD and
the andronate groups had a lower insulin sensitivity
index (-4.6 ± 0.10 and -4.35 ± 0.15, respectively) than
the control group (-3.87 ± 0.10, P <0.05) (Figure 8).

Profiles of serum hormone levels

Although FSH levels were not affected, the LH levels
were increased in the andronate-treated groups. Conse-
quently, the LH/FSH ratio was elevated in the treated
group, mirroring the changes observed in human PCOS.
Furthermore, testosterone levels were significantly aug-
mented in the andronate + HFD group, indicating that

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Figure 5 The main stages of the reproductive cycle in the control rats. Swab smears (unstained) shown with the original microscope
magnification of 100x. Afstrus stage: large cornified cells in clumps. B: Metestrus stage: large numbers of leucocytes with smaller numbers of
non-nucleated epithelial cells (note characteristic clumping together of two cell types at the center-right). C: Diestrus stage: predominantly
leucocytes with a small number of epithelial and cornified cells. D: Proestrus stage:epithelial cells are mostly rounded but some cells shows early
stages of cornification of approaching estrus.

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hyperandrogenism was present in this animal model
(Table 1).

Plasma lipid profile

Disturbances in the plasma lipid profiles were appar-
ent (Table 2). There were increases in plasma concen-
trations of TC (total cholesterol), TG (triglycerides),
and LDL-C (high density lipoprotein-cholesterol) in
andronate-treated rats, and the plasma concentrations
of these molecules were further elevated in the andro-
nate + HFD group. Plasma HDL-C (high density lipo-
protein -cholesterol) levels also tended to be elevated
in the two groups, but the difference was not
significant.

Rates of glucose appearance (glucose Ra),
gluconeogenesis (GNG), rates of glycerol appearance
(glycerol Ra)

The glucose Ra was significantly increased in the andro-
nate-treated groups, while that of glycerol was not
affected. Fractional gluconeogenesis was most elevated
in the andronate + HFD group, indicating that the com-
bination of andronate and high-fat diet can increase
body glucose by accelerating gluconeogenesis from gly-
cerol (Table 3).

Discussion

Metabolism Syndrome (MS) is estimated to affect
approximately 40% of women with PCOS, leading to

Figure 7 Body hair in the three rats groups. At 10 weeks of age, pictures were taken of the 3 groups. A:control group; B:andronate group; C:
andronate with HFD group.

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-E-|

1

I

1 UTTTTl'J

CI Aj'LltLjy.'U

j M Buritam-fc+Hr

1 . r*rl

FBG&ikdWO JTHS fmOJLJ HOHA-CF.

Figure 8 FBG, FINS, HOMA-IR and ISI in the 3 groups. Tail blood was obtained after an overnight fast in 10-week-old rats to assess fasting
blood glucose (FBG), fasting plasma insulin (FINS), homeostasis model-insulin resistance (HOMA-IR) and insulin sensitivity index(ISI) in 3 groups.

increased prevalence of hypertension, dyslipidemia, and
abnormal glucose metabolism [11]. We here demon-
strate that andronate with HFD rats replicate both the
ovarian and the metabolic syndrome features of human
PCOS, including PCO morphology, irregular cycles,
increased body weight, hyperlipidemia, and insulin
resistance.

It has been suggested that adolescent obesity is corre-
lated with insulin resistance (IR), dyslipidemia, and
PCOS related ovulatory dysfunction [12]. In the present
study, obesity was observed in andronate-treated groups;
however, there was no significant difference between the
groups fed with normal or high-fat diets. These results
were consistant to our previous study of castrated male
rats [13], and is likely due to failed adaptation to HFD.

Up to 70% of women with PCOS are also insulin
resistant (IR), and the prevalence of DM in women with

Table 1 FSH, LH, LH/FSH and T of andronate treated and
control groups

Group

Control

Andronate

Andronate+high-fat
diet

FSH(mlU/

0.018 ±

0.017 ± 0.0082

0.017 ± 0.0052

ml)

0.0075

LH(mlU/ml)

0.045 ±

0.058 ±

0.053 ± 0.01 6 a

0.0014

0.0021 a

LH/FSH

2.61 ± 0.61

3.75 ± 1.1 7 a

3.25 ± 0.52 a

T(ng/ml)

2.37 ± 0.23

14.33 ± 0.92 b

16.35 ± 1.55 b

Values expressed as mean ± SD, a: p < 0.05, b: p < 0.01

PCOS is 10% [14]. IR is a state of impaired metabolic
response to insulin as characterized by the American
Diabetes Association (ADA) [15]. The probable mechan-
isms of insulin-related reproductive abnormalities
include excessive LH secretion, abnormalities of ovarian
steroidogenesis, and abnormal glucose uptake in PCOS
[16]. Although some women with PCOS initially exhibit
normal glucose metabolism, the conversion rate of
abnormal glucose metabolism in 3 years is 25% [17].

This study describes an apparent insulin resistance
with increased HOMA-IR and decreased ISI in the
andronate-treated group fed with HFD, though blood
glucose (BG) of the three groups remains within normal
range. Therefore, although the glucose metabolism
abnormalities did not affect the blood glucose levels, the
potential insulin resistance might have already existed.
Thus, blood glucose level alone can not predict meta-
bolic risk in PCOS, and the precursor states of insulin

Table 2 TG, TC, HDL-C and LDL-C of andronate treated

and control groups

Group

Control

Andronate

Andronate +high-fat

TG(mmol/L)

0.80 ± 0.1 1

1.06 ± 0.1 3 a

1.24 ± 0.1 7 b

TC(mmol/L)

1.51 ± 0.15

1.88 ± 0.1 5 a

2.32 ± 0.1 6 b

HDL-C(mmol/L)

0.56 ± 0.10

0.57 ± 0.09

0.61 ± 0.10

LDL-C(mmol/L)

0.46 ±0.10

0.50 ± 0.09

0.62 ± 0.05 a

Values expressed as mean ± SD, a: p < 0.05, b: p < 0.01

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Table 3 Glucose, glycerol Ra, GNG, MPE of U-C13-glycerol
and 1,2,3-C13-glucose j n andronate treated and control
groups

Group Control Andronate Andronate +HF

Glucose Ra (umol/kg/min)

52.7 ±

9.6

73.8 ±

12.4 a

79.4 :

b 11.5 a

Glycerol Ra(umol/kg/min)

17.3 ±

6.2

17.8 ±

3.4

16.5 :

b 4.5

GNG(%)

13.4 ±

1.5

16.7 ±

2.3 a

18.5 :

b 4.7 a

Mpe of U-C13-glycerol

4.87 ±

0.68

5.03 ±

0.63 a

5.34 :

b 0.79 b

Mpe of 1,2,3-C13-glucose

0.63 ±

0.12

0.82 ±

0.1 1 a

0.97 :

b 0.20 a

Values expressed as mean ±

SD, a: p ■

< 0.05

, b: p < 0.01

abnormalities may predict the risk of DM well before
BG abnormalities arise [18].

The abnormalities of glucose and lipid metabolism
were evaluated with stable isotopes. The results sug-
gested a higher rate of glucose appearance (Ra of glu-
cose) and gluconeogenesis (GNG) in andronate + HFD
rats; though the rate of glycerol appearance (Ra of gly-
cerol) showed no differences between the three groups.
Ra of glycerol provides a good measurement of whole-
body lipolysis during fasting. Because triacylglycerol is
broken down into glycerol and three fatty acids, the
amount of fatty acids released into circulation is three
times the rate of glycerol production. Therefore,
although this study showed no significant difference in
the Ra of glycerol among the three groups, the lipid pro-
files change significantly in the andronate-treated goups.
The increased Ra of glucose and GNG in combination
with the unaffected BG indicate that the dynamics of
glucose metabolism have been actively initiated in the
early state, which maybe compensated increased to
antagonist the elevated plasma insulin.

For the past several years, a number of PCOS rat
models have been used to study the etiology and patho-
physiology of PCOS. However, all of these methods
have their limitations. As PCOS can be induced with
estradiol valerate, it has been found that estradiol vale-
rate results in acyclicity and PCOS-like ovarian mor-
phology but does not cause the typical metabolic
disturbances of human PCOS [19]. In another promising
rat model, PCOS is induced by letrozole, an aromatase
inhibitor that blocks the conversion of testosterone to
estradial. While this model exhibits morphological simi-
larities to human PCOS, it does not decrease insulin
sensitivity despite increased testosterone concentrations
[20]. After continuous exposure to dihydrotestosterone
(DHT), rats develop PCOS characteristics, including
increased number of apoptotic follicles, obesity, and
insulin resistance. Thus, the DHT model is generally
preferable for studies of both ovarian and metabolic fea-
tures. However, in this model, the concentration of tes-
tosterone is low, and the mechanism of the endogenous
production of androgens or testosterone is likely to be

reduced by DHT treatment [21]. It has been strongly
suggested that high-fat diet can induce liver insulin
resistance [22] and that rats fed with HFD for 6 weeks
are notably hyperinsulinemia. Therefore, andronate
combined with high-fat diet to establish a rat model
that manifests increased similarities in the ovary and
metabolic features of PCOS.

The relationship between hyperandrogenism and IR is
significant [23]. Many human research studies have
demonstrated the high degree of this correlation [24].
Furthermore, the risk of metabolic syndrome in females
with PCOS is highly correlated with increased testoster-
one concentrations [25]. A recent review demonstrated
that women with elevated T concentrations have a
higher risk of developing type 2 diabetes mellitus [26].
Animal model have also shown that prenatal testoster-
one exposure can induce IR in early postnatal life [27],
which was confirmed in the present study.

Conclusions

A rat model of PCOS was successfully established with
features of polycystic ovaries, obesity, irregular cycles of
vaginal smear, increased plasma insulin levels, decreased
insulin sensitivity, hyperandrogenism, and increased LH
concentrations. This animal model also exhibited signifi-
cant increase in glucose Ra, rates of GNG, and lipid
profiles despite having normal blood glucose levels.
Therefore, we propose that IR, glucose Ra, GNG rates
and lipid metabolism may be potential treatment targets
for early-stage PCOS. Further studies are needed to
evaluate the effects of these treatments on PCOS and to
elucidate the complexity of PCOS etiology and
pathophysiology.

Abbreviations

DM: Diabetes mellitus; PCOS: Polycystic ovary syndrome; HFD: High-fat diet;
Ra of glucose: Rate of glucose appearance; GNG: Gluconeogenesis; Ra of
glycerol: Rate of glycerol appearance; IR: Insulin resistance; MPAIA: Magnetic
particle antibody immunoassay; FSH: follicle-Stimutating hormone; LH:
Luteinizing hormone; T: Testosterone; CMIA: Chemiluminescent microparticle
immunoassay; GC: Gas chromatography; MPE: Mole percent excess; SD:
Standard deviation; FBG: Fasting blood glucose; FINS: Fasting plasma insulin;
HOMA-IR: Homeostasis model-insulin resistance; ISI: Insulin sensitivity index;
TC: Total cholesterol; TG: Triglycerides; LDL-C: High density lipoprotein-
cholesterol; HDL-C: High density lipoprotein-cholesterol; MS: Metabolism
Syndrome; ADA: American diabetes association; DHT: Dihydrotestosterone.

Acknowledgements

We would like to thank Professor Zeng-Kui Guo has given us a lot of help in
isotope analysis. Funding: This study was supported by the National Nature
Science Foundation of China (NSFC81 070677). We have made language
corrections by MedSci. Certificate code: 0612-4928-66B8-9D5A-9B5A.

Authors' contributions

H-LZ has supervised the entire work on the animals, done the isotope
analysis and wrote the manuscript. HW has helped the animal catheterized.
HX has done the statistical analysis of data and PW has done the animal
model construction including ovary morphology HE staining and vaginal
smears. F-ZX has done the lipid assays and YC has done the insulin assays.

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Y-LL contributed to the concept of the study and has supervised all the
work. All authors read and approved the final manuscript.

Authors' information

Hua-Ling Zhai during the experiment was PhD student. She is now
attending physician at Endocrinology and Metabolism Research Institute and
Department of Endocrinology and Metabolism, Shanghai Ninth People's
Hospital Affiliated Shanghai Jiaotong University School of Medicine. Hui Wu,
Hui Xu, Yi Chen and Pan weng are all postgraduate students. Fang-Zhen Xia
is a permanent research worker. Ying-Li Lu is professor and director of
endocrinology department in Shanghai Ninth People's Hospital Affiliated
Shanghai Jiaotong University School of Medicine.

Competing interests

The authors declare that they have no competing interests.

Received: 29 October 2011 Accepted: 25 January 2012
Published: 25 January 2012

References

1. Ehrmann DA: Polycystic ovary syndrome. N Engl J Med 2005,
352(1 2):1 223-1 236.

2. Norman RJ, Dewailly D, Legro RS, Hickey TE: Polycystic ovary syndrome.
Lancet 2007, 370:685-697.

3. Barber TM, McCarthy Ml, Wass JA, Franks S: Obesity and polycystic ovary
syndrome. Clin Endocrinol (Oxf) 2006, 65:137-145.

4. Nelson VL, Qin K-N, Rosenfield RL, Wood JR, Penning TM, Legro RS,
Strauss JF, McAllister JM: The biochemical basis for increased testosterone
production in theca cells propagated from patients with polycystic
ovary syndrome. J Clin Endocrinol Metob 2001, 86:5925-5933.

5. Soule SG: Neuroendocrinology of the polycystic ovary syndrome. Boillieres
Clin Endocrinol Metob 1996, 10(2)205-219.

6. Legro RS, Kunselman AR, Dodson WC, Dunaif A: Prevalence and predictors
of risk for type 2 diabetes mellitus and impaired glucose tolerance in
polycystic ovary syndrome: a prospective, controlled study in 254
affected women. J Clin Endocrinol Metob 1999, 84(1 ):1 65-1 69.

7. Dunaif A, Finegood DT: Beta-cell dysfunction independent of obesity and
glucose intolerance in the polycystic ovary syndrome. J Clin Endocrinol
Metob 1996, 81(3):942-947.

8. Aden P, Quereda F, Matallin P, Villarroya E, Lopez-Fernandez JA, Aden M,
Mauri M, Alfayate R: Insulin, androgens, and obesity in women with and
without polycystic ovary syndrome: a heterogeneous group of disorders.
FertilSteril 1999, 72:32-40.

9. Traub ML: Assessing and treating insulin resistance in women with
polycystic ovarian syndrome. World J Diabetes 201 1, 2(3):33-40.

10. Radavelli-Bagatini S, Blair AR, Proietto J, Spritzer PM, Andrikopoulos S: The
New Zealand obese mouse model of obesity insulin resistance and poor
breeding performance: evaluation of ovarian structure and function. J
Endocrinol 201 1, 209(3):307-315.

11. El-Mazny A, Abou-Salem N, El-Sherbiny W, El-Mazny A: Insulin resistance,
dyslipidemia, and metabolic syndrome in women with polycystic ovary
syndrome. Int J Gynaecol Obstet 2010, 109:239-241.

12. Bougie D, Zunquin G, Sesboue B, Sabatier JP: Relationships of
cardiorespiratory fitness with metabolic risk factors, inflammation, and
liver transaminases in overweight youths. Int J Pediatr 2010, 2010:1-5.

13. Lu YL, Jiang BR, Xia FZ, Zhai HL, Chen Y, Yu J, Zhao LJ, Wang NJ, Qiao J,
Yang LZ: Changes of pituitary and penile structure in male adult rats
following castration and high-fat diet. J Endocrinol Invest 201 1,
34(2):111-116.

14. Freeman R, Pollack R, Rosenbloom E: Assessing impaired glucose
tolerance and insulin resistance in Polycystic Ovarian Syndrome with a
muffin test: alternative to glucose tolerance test. Endocr Pract 2010,
16(5):810-817.

15. American Diabetes Association: Consensus development conference on
insulin resistance. Diabetes Care 1998, 21:310-314.

16. Baptiste CG, Battista MC, Trottier A, Baillargeon JP: Insulin and
hyperandrogenism in women with polycystic ovary syndrome. J Steroid
Biochem Mol Biol 2010, 122:42-52.

17. Pesant MH, Baillargeon JP: Clinically useful predictors of conversion to
abnormal glucose tolerance in women with polycystic ovary syndrome.
FertilSteril 201 1,95:210-215.

19.

20.

22.

23.

24.

25.

26.

27.

Ishihara M, Inoue I, Kawagoe T, Shimatani Y, Kurisu S, Nakama Y, Kagawa E,
Dai K, Ootani T, Ikenaga H, Morimoto Y, Ejiri K, Oda N: Glucometabolic
responses during glucose tolerance test: a comparison between known
diabetes and newly detected diabetes after acute myocardial infarction.

Int J Cardiol 2011, 152(1)78-82.

Manni L, Cajander S, Lundeberg T, Naylor AS, Aloe L, Holmang A,
Jonsdottir IH, Stener-Victorin E: Effect of exercise on ovarian morphology
and expression of nerve growth factor and alpha(l)- and beta--
adrenergic receptors in rats with steroid-induced polycystic ovaries. J

Neuroendocrinol 2005, 1 7(1 2):846-858.

Kafali H, Iriadam M, Ozardali I, Demir N: Letrozole-induced polycystic
ovaries in the rat: a new model for cystic ovarian disease. Arch Med Res

2004, 35(2):103-108.

Manneras L, Cajander S, Holmang A, Seleskovic Z, Lystig T, Lonn M, Stener-
Victorin E: A new rat model exhibiting both ovarian and metabolic
characteristics of polycystic ovary syndrome. Endocrinology 2007,
148(8)3781-3791.

Minassian C, Tarpin S, Mithieux G: Role of Glucose-6 Phosphatase,
Glucokinase, and Glucose-6 Phosphate in Liver Insulin Resistance and Its
Correction by Metformin. Biochem Pharmacol 1998, 55:1213-1219.
Aden P, Quereda F, Matallin P, Villarroya E, Lopez-Fernandez JA, Aden M,
Mauri M, Alfayate R: Insulin, androgens, and obesity in women with and
without polycystic ovary syndrome: a heterogeneous group of disorders.
FertilSteril 1999, 72:32-40.

Luque-Ramirez M, Alpanes M, Escobar-Morreale HF: The determinants of
insulin sensitivity, (3-cell function, and glucose tolerance are different in
patients with polycystic ovary syndrome than in women who do not
have hyperandrogenism. Fertil Steril 2010, 94:2214-2221.
Coviello AD, Legro RS, Dunaif A: Adolescent girls with polycystic ovary
syndrome have an increased risk of the metabolic syndrome associated
with increasing androgen levels independent of obesity and insulin
resistance. J Clin Endocrinol Metab 2006, 91:492-497.
Ding EL, Song Y, Malik VS, Liu S: Sex differences of endogenous sex
hormones and risk of type 2 diabetes: a systematic review and meta-
analysis. JAMA 2006, 295:1288-1299.

Padmanabhan V, Veiga-Lopez A, Abbott DH, Recabarren SE, Herkimer C:
Developmental programming: impact of prenatal testosterone excess
and postnatal weight gain on insulin sensitivity index and transfer of
traits to offspring of overweight females. Endocrinology 2010, 151:595-605.

doi:1 0.1 1 86/1 477-7827-1 0-5

Cite this article as: Zhai et al:. Trace glucose and lipid metabolism in
high androgen and high-fat diet induced polycystic ovary syndrome
rats. Reproductive Biology and Endocrinology 2012 10:5.

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